Numerous methods have been used to sequence nucleic acid molecules. The traditional Maxam-Gilbert chemical degradation method involves the chemical-specific cleavage of DNA (Maxam and Gilbert, Proc. Natl. Acad. Sci., USA 74:560, 1977). In this method, radio-labeled DNA molecules are incubated in four separate reaction mixtures, each of which partially cleaves the DNA at one or two nucleotides of a specific identity (G, A+G, C or C+T). The resulting DNA fragments are separated by polyacrylamide gel electrophoresis, with each of the four reactions fractionated in a separate lane of the gel. The DNA sequence is determined after autoradiography by observing the macromolecular separation of the fragments in the four lanes of the gel.
The Sanger dideoxy chain termination method involves generating DNA molecules of differing lengths by enzymatic extension of a synthetic primer, using DNA polymerase and a mixture of deoxy- and dideoxy-nucleoside triphosphates (Sanger et al., Proc. Natl. Acad. Sci., USA 74:5463, 1977). The reactions are separated in four parallel lanes on polyacrylamide gels and the sequence determined after autoradiography.
The use of fluorescent nucleotides has eliminated the need for radioactive nucleotides, and provides a means to automate DNA sequencing (for example see U.S. Pat. No. 5,124,247 to Ansorge, U.S. Pat. No. 5,242,796 to Prober et al., U.S. Pat. No. 5,306,618 to Prober et al., U.S. Pat. No. 5,360,523 to Middendorf et al., U.S. Pat. No. 5,556,790 to Pettit, and U.S. Pat. No. 5,821,058 to Smith et al.). However, methods that use fluorophores generally still require gels or capillary electrophoresis, and thus are slow and macroscopic.
Another potential obstacle with using fluorescently labeled dNTPs is that no one has been able to synthesize a fully fluorescently labeled DNA molecule. Therefore, sequencing methods that permit the synthesis of the complementary nucleic acid strand are still needed.